Mast cells play a pivotal role in the pathogenesis of asthma and other allergic diseases. These reactions are generally initiated by antigen-dependent aggregation of the high affinity IgE receptor (Fc-epsilon-RI) expressed on the cell surface and subsequent release of pro-inflammatory mediators (e.g. histamine, eicosanoids, proteases and cytokines). We wish to identify disease states and clinical populations where hyper-responsive and hypo-responsive mast cells exist and to identify specific signaling defects responsible for these phenotypes. In addition to the Fc-epsilon-RI, there is an increasing appreciation that other receptors (and other stimuli) may profoundly influence antigen-mediated degranulation. Activating polymorphisms/mutations in, and alternatively spliced forms of, receptors and/or signaling proteins may further modulate these responses. Such polymorphisms associated with disease states, like anaphylaxis, atopy, and mastocytosis may be manifested by exacerbated mast cell-dependent physiology. We wish therefore to also explore the role of other mast cell receptors in disease states and how polymorphisms or alternatively spliced variants of receptors or signaling proteins may produce a hyperactive phenotype. Finally, we wish to explore potential approaches for inhibiting these responses. The following observations were made since the last report: i. Mast cell phenotype identified in idiopathic anaphylaxis Mast cells, generated from peripheral blood progenitors from a subset of idiopathic anaphylaxis patients were observed to have a hyper-proliferative, hyper-responsive phenotype. These features may be linked to exaggerated antigen-mediated signaling processes. Studies are underway to identify the genetic basis for this phenotype. ii. Human mast cells display differential phenotypes in their ability to synergistically respond to antigen and G protein-coupled receptor (GPCR) agonists. PGE2 is produced in inflamed tissues, and, in mouse BMMCs, has been found to markedly enhance mast cell activation. Thus, PGE2 may contibute to activation of mast cells in disease states. In investigating the ability of PGE2 to potentiate antigen-mediated activation of human mast cells, we observed that mast cells generated from peripheral blood of only half of the randomly selected donors examined were responsive to PGE2 or to the EP3 agonist sulprostone with regards to degranulation and cytokine generation. We determined that these differences were not due to variation in receptor expression but were linked to a lack of ability of PGE2 to potentiate the necessary antigen-mediated calcium signal in the non-responders. Our observations may have important implications for mast cell-driven disease, in that the extent to which mast cells may trigger in specific atopic individuals, in part, may be a factor of their ability to respond, or not, to circulating co-activators. Thus, our findings may serve as a basis for investigating the potential roles of mast cell co-activators and alterations in their signaling capapcity in the etiology of mast cell-driven disorders. iii. Determination of the role of TRPM8 in mast cell activation in cold-induced urticaria. The cold-sensing calcium channel TRPM8 has been implicated in the etiology of cold-induced urticaria. Nevertheless, the role of TRPM8 in this condition has not been adequately established. We thus investigated the potential role(s) of TRPM8 in mast cell activation and whether mutations in the TRPM8 channel could account for the manifestations of the disease. We observed no evidence of TRPM8 expression or function in human mast cells, nor meaningful mutations in TRPM8 in the serum of cold-induced urticaria patients. Furthermore, no defects were observed in mast cells generated from the bone marrow of TRPM8-deficient mice. Taken together, these data demonstrate that other mechanisms acount for the activation of mast cells associated with cold-induced urticaria.